Background: Identifying developmental processes regulated by Notch1 can be addressed in part by characterizing mice with graded levels of Notch1 signaling strength. Here we examine development in embryos expressing various combinations of Notch1 mutant alleles. Mice homozygous for the hypomorphic Notch1(12f) allele, which removes the single O-fucose glycan in epidermal growth factor-like repeat 12 (EGF12) of the Notch1 ligand binding domain (lbd), exhibit reduced growth after weaning and defective T cell development. Mice homozygous for the inactive Notch1(lbd) allele express Notch1 missing an aproximately 20 kDa internal segment including the canonical Notch1 ligand binding domain, and die at embryonic day approximately E9.5. The embryonic and vascular phenotypes of compound heterozygous Notch1(12f/lbd) embryos were compared with Notch1+/12f, Notch1(12f/12f), and Notch1(lbd/lbd) embryos. Embryonic stem (ES) cells derived from these embryos were also examined in Notch signaling assays. While Notch1 signaling was stronger in Notch1(12f/lbd) compound heterozygotes compared to Notch1(lbd/lbd) embryos and ES cells, Notch1 signaling was even stronger in embryos carrying Notch1(12f) and a Notch1 allele.

Results: Mouse embryos expressing the hypomorphic Notch1(12f) allele, in combination with the inactive Notch1(lbd) allele which lacks the Notch1 ligand binding domain, died at approximately E11.5-12.5. Notch1(12f/lbd) ES cells signaled less well than Notch1(12f/12f) ES cells but more strongly than Notch1lbd/lbd ES cells. However, vascular defects in Notch1(12f/lbd) yolk sac were severe and similar to Notch1(lbd/lbd) yolk sac. By contrast, vascular disorganization was milder in Notch1(12f/lbd) compared to Notch1(lbd/lbd) embryos. The expression of Notch1 target genes was low in Notch1(12f/lbd) yolk sac and embryo head, whereas Vegf and Vegfr2 transcripts were increased. The severity of the compound heterozygous Notch1(12f/lbd) yolk sac phenotype suggested that the allelic products may functionally interact. By contrast, compound heterozygotes with Notch112f in combination with a Notch1 allele (Notch1(tm1Con)) were capable of surviving to birth.

Conclusions: Notch1 signaling in Notch1(12f/lbd) compound heterozygous embryos is more defective than in compound heterozygotes expressing a hypomorphic Notch1(12f) allele and a Notch1 allele. The data suggest that the gene products Notch1(lbd) and Notch1(12f) interact to reduce the activity of Notch1(12f).

Figure 5: Real-time PCR of vasculogenic and Notch target genes in Notch112f/lbd yolk sac and embryo. Total RNA extracted from E10.5 yolk sac or embryonic head was reverse-transcibed and subjected to real-time PCR. Numbers of transcripts were normalized to β-actin, and the average relative expression of Notch1+/12f samples was set to 1. (A-F) Relative expression of Pecam1, Vegf, Vegfr2, Hes5, Hey1, and Hey2 as marked. Bars represent SEM (n = 6). The two-tailed Student's t test was used in control versus mutant yolk sac and embryo head comparisons; a one-tailed Student's t test was used in mutant yolk sac versus mutant embryo head comparisons; * p < 0.05; ** p < 0.01

Mentions:
Whereas vascularization was severly affected in both yolk sac and embryo of Notch1lbd/lbd embryos, only the yolk sac of Notch112f/lbd compound heterozygous embryos exhibited extremely defective vascularization. To investigate further, the expression of vasculogensis-related and Notch1 target genes was examined by real-time PCR using total RNA isolated from E10.5 Notch112f/lbd and Notch1+/12f yolk sacs and embryo heads. The relative expression levels of Pecam1 and Vegf were increased in Notch112f/lbd yolk sacs and embryos, and Vegfr2 expression was increased in Notch112f/lbd embryo heads (Fig. 5A-C). Therefore loss of Notch1 signaling upregulated transcription of the Pecam1, Vegf and Vegfr2 genes. Interestingly, the increased expression of Vegf and Vegfr2 was greater in Notch112f/lbd embryos, consistent with the relative strength of Notch1 signaling being greater in yolk sac. Expression of the Notch1 target genes Hes5, Hey1 and Hey2 was reduced in Notch112f/lbd yolk sac (Fig. 5D-F), but the level of Hes1 transcripts was not changed (data not shown). In embryos, only the expression of Hes5 was significantly reduced compared to control. The expression of Ang1, Tie2 and Ephrin-B2 which are involved in angiogenesis, as well the expression of Notch1 itself, were not changed in Notch112f/lbd yolk sac or embryos (data not shown). The fact that the increase in Vegf and Vegfr2 transcripts was more in embryo head than yolk sac (418% vs 170% for Vegf; 227% vs. 148% for Vegfr2; Fig. 5B and 6C), and the fact that the reduction in Notch target gene expression was greater in yolk sac than embryo head, correlated generally with Notch1 signal strength and the greater severity of vascularization defects in yolk sac versus embryo head.

Figure 5: Real-time PCR of vasculogenic and Notch target genes in Notch112f/lbd yolk sac and embryo. Total RNA extracted from E10.5 yolk sac or embryonic head was reverse-transcibed and subjected to real-time PCR. Numbers of transcripts were normalized to β-actin, and the average relative expression of Notch1+/12f samples was set to 1. (A-F) Relative expression of Pecam1, Vegf, Vegfr2, Hes5, Hey1, and Hey2 as marked. Bars represent SEM (n = 6). The two-tailed Student's t test was used in control versus mutant yolk sac and embryo head comparisons; a one-tailed Student's t test was used in mutant yolk sac versus mutant embryo head comparisons; * p < 0.05; ** p < 0.01

Mentions:
Whereas vascularization was severly affected in both yolk sac and embryo of Notch1lbd/lbd embryos, only the yolk sac of Notch112f/lbd compound heterozygous embryos exhibited extremely defective vascularization. To investigate further, the expression of vasculogensis-related and Notch1 target genes was examined by real-time PCR using total RNA isolated from E10.5 Notch112f/lbd and Notch1+/12f yolk sacs and embryo heads. The relative expression levels of Pecam1 and Vegf were increased in Notch112f/lbd yolk sacs and embryos, and Vegfr2 expression was increased in Notch112f/lbd embryo heads (Fig. 5A-C). Therefore loss of Notch1 signaling upregulated transcription of the Pecam1, Vegf and Vegfr2 genes. Interestingly, the increased expression of Vegf and Vegfr2 was greater in Notch112f/lbd embryos, consistent with the relative strength of Notch1 signaling being greater in yolk sac. Expression of the Notch1 target genes Hes5, Hey1 and Hey2 was reduced in Notch112f/lbd yolk sac (Fig. 5D-F), but the level of Hes1 transcripts was not changed (data not shown). In embryos, only the expression of Hes5 was significantly reduced compared to control. The expression of Ang1, Tie2 and Ephrin-B2 which are involved in angiogenesis, as well the expression of Notch1 itself, were not changed in Notch112f/lbd yolk sac or embryos (data not shown). The fact that the increase in Vegf and Vegfr2 transcripts was more in embryo head than yolk sac (418% vs 170% for Vegf; 227% vs. 148% for Vegfr2; Fig. 5B and 6C), and the fact that the reduction in Notch target gene expression was greater in yolk sac than embryo head, correlated generally with Notch1 signal strength and the greater severity of vascularization defects in yolk sac versus embryo head.

Bottom Line:
The embryonic and vascular phenotypes of compound heterozygous Notch1(12f/lbd) embryos were compared with Notch1+/12f, Notch1(12f/12f), and Notch1(lbd/lbd) embryos.By contrast, vascular disorganization was milder in Notch1(12f/lbd) compared to Notch1(lbd/lbd) embryos.The data suggest that the gene products Notch1(lbd) and Notch1(12f) interact to reduce the activity of Notch1(12f).

Background: Identifying developmental processes regulated by Notch1 can be addressed in part by characterizing mice with graded levels of Notch1 signaling strength. Here we examine development in embryos expressing various combinations of Notch1 mutant alleles. Mice homozygous for the hypomorphic Notch1(12f) allele, which removes the single O-fucose glycan in epidermal growth factor-like repeat 12 (EGF12) of the Notch1 ligand binding domain (lbd), exhibit reduced growth after weaning and defective T cell development. Mice homozygous for the inactive Notch1(lbd) allele express Notch1 missing an aproximately 20 kDa internal segment including the canonical Notch1 ligand binding domain, and die at embryonic day approximately E9.5. The embryonic and vascular phenotypes of compound heterozygous Notch1(12f/lbd) embryos were compared with Notch1+/12f, Notch1(12f/12f), and Notch1(lbd/lbd) embryos. Embryonic stem (ES) cells derived from these embryos were also examined in Notch signaling assays. While Notch1 signaling was stronger in Notch1(12f/lbd) compound heterozygotes compared to Notch1(lbd/lbd) embryos and ES cells, Notch1 signaling was even stronger in embryos carrying Notch1(12f) and a Notch1 allele.

Results: Mouse embryos expressing the hypomorphic Notch1(12f) allele, in combination with the inactive Notch1(lbd) allele which lacks the Notch1 ligand binding domain, died at approximately E11.5-12.5. Notch1(12f/lbd) ES cells signaled less well than Notch1(12f/12f) ES cells but more strongly than Notch1lbd/lbd ES cells. However, vascular defects in Notch1(12f/lbd) yolk sac were severe and similar to Notch1(lbd/lbd) yolk sac. By contrast, vascular disorganization was milder in Notch1(12f/lbd) compared to Notch1(lbd/lbd) embryos. The expression of Notch1 target genes was low in Notch1(12f/lbd) yolk sac and embryo head, whereas Vegf and Vegfr2 transcripts were increased. The severity of the compound heterozygous Notch1(12f/lbd) yolk sac phenotype suggested that the allelic products may functionally interact. By contrast, compound heterozygotes with Notch112f in combination with a Notch1 allele (Notch1(tm1Con)) were capable of surviving to birth.

Conclusions: Notch1 signaling in Notch1(12f/lbd) compound heterozygous embryos is more defective than in compound heterozygotes expressing a hypomorphic Notch1(12f) allele and a Notch1 allele. The data suggest that the gene products Notch1(lbd) and Notch1(12f) interact to reduce the activity of Notch1(12f).